Macrophages (MF) are essential for immunity against pathogens, tissue homeostasis and immune regulation. Helminth infections, allergic reactions and tissue injury can induce the differentiation of alternatively activated macrophages (AAMF), which are important in promoting tissue remodeling, wound repair, T helper 2 (TH2) differentiation and parasite clearance. The differentiation process and cellular precursors of AAMF remains poorly understood. Recently, two functionally distinct subsets of monocytes and their properties have been described; (1) Ly-6C+ inflammatory monocytes and (2) Ly6C- resident monocytes. While Ly6C- monocytes populate normal tissues, they have also been shown to patrol blood vessels and extravasate rapidly into inflamed or infected tissues to promote the resolution of inflammation. Ly6C- cells are also CX3CR1-GFPhi in a CX3CR1-GFP reporter mouse and have been best characterized in models of myocardial infarction and bacterial infection. In both models, extravasated Ly6C-, CX3CR1-GFPhi monocytes have characteristics of AAMF and can promote tissue remodeling, wound healing and immune modulation. We have previously shown that helminth infection potently induces recruitment of AAMF. Others have shown that the recruitment of AAMF is critical in protecting S. mansoni infected mice from acute immunopathology in response to the eggs. We have also recently shown that sterile tissue injury can induce recruitment of AAMF in the absence of infection, through a T cell independent innate immune pathway. We have now conducted preliminary flow cytometry, confocal microscopy and intra-vital imaging studies of the liver granulomas of S. mansoni infected CX3CR1-GFP/+ mice, which suggest that AAMF are CX3CR1-GFPhi and may arise from the Ly6C-, CX3CR1-GFPhi monocytes that are patrolling the sinusoidal vessels. In this proposal, we propose to use intra-vital microscopy to observe T cell-macrophage interactions in the liver granulomas of S. mansoni infected mice. Specifically, we propose to test the hypothesis that AAMF recruited by S. mansoni eggs differentiate from CX3CR1-GFPhi, Ly6C- monocytes. As a secondary hypothesis, we propose that CD4+ T cells may play a role in recruiting or maintaining CX3CR1-GFP+ cells into the granulomas to differentiate into AAMF during chronic infection. Therefore, our specific aims are: (1) to visualize the dynamics of monocyte recruitment and macrophage differentiation in liver granulomas using the CX3CR1- GFP reporter mice; (2) to determine the role of CD4+ TH2 cells in the recruitment of CX3CR1-GFPhi cells by S. mansoni eggs in the liver granulomas. These studies will improve our understanding of monocyte recruitment and macrophage differentiation under Th2 conditions and may provide us with a framework for new interventional therapies to regulate pathogenic inflammatory Th2 responses. PUBLIC HEALTH RELEVANCE: Macrophages activated under T helper type 2 conditions are important in wound healing, allergic reactions and parasite infections. How these cells are recruited into the tissues from monocytes in the blood is not clear. The goal of this project is to identify where these macrophages come from in order to design interventional strategies that could help regulate the inflammatory process during a type 2 response.